Tumor-derived DNA is released into the bloodstream from dying cancer cells in patients with various types of malignancies. Such circulating tumor DNA (ctDNA) is showing excellent promise as a non-invasive cancer biomarker. However, an assay that is capable of exploiting ctDNA for early cancer detection presents several challenges. In the bloodstream, ctDNA can be distinguished from normal background DNA based on the presence of tumor-specific mutations. However, mutant ctDNA is usually only present in small amounts, having been previously reported to comprise an average of 0.2% of total plasma DNA (Diehl et al., Nat Med. 2008; 14: 985-990). If variant DNA sequences are low in abundance, detecting and quantifying these variants can be more challenging. Small amounts of mutation-harboring ctDNA can be obscured by a relative excess of background wild-type plasma DNA. Thus, an assay with extremely high detection sensitivity is required.
There is a need for a method that is able to detect and quantify rare variant sequences to detect cancers in situations where the amount of DNA in a given sample is limited. Unlike existing approaches, a test should be able to evaluate an entire panel of mutation-prone regions without needing to divide DNA samples into separate reactions (which could reduce detection sensitivity by providing fewer template DNA copies per reaction). Methods and compositions are described herein that provide a multiplex assay to detect minute amounts of ctDNA and address the current deficiencies to assay ctDNA.